Bovine Aortic Endothelial Cells In Culture Research Articles

570-P: Osteoprotegerin Induces Endothelial Dysfunction and Is Associated with Vascular Complications In Type 2 Diabetes

Novel markers of vascular disease in diabetes could help identify new disease pathways and enhance risk stratification. Osteoprotegerin (OPG) and osteopontin (OPN) are key molecules involved in bone and vascular calcification processes, both compromised in diabetes. To evaluate whether OPG and/or OPN are associated with diabetic vascular complications, we measured their serum concentrations in 995 type 2 diabetes subjects enrolled in the Sapienza University Mortality and Morbidity Event Rate (SUMMER) Study. Any association with history of cardiovascular disease (CVD), diabetic retinopathy (DR) and reduced eGFR were examined with ANOVA, correcting for age, gender, HbA1c and lipid profile. Among the population, 15.7% had CVD, 18.4% had DR, 12.0% had an eGFR between 45-59 ml/min/1.73m2 and 7.5% had an eGFR <45 ml/min/1.73m2. Serum OPG, but OPN, concentrations stratified according to the presence of CVD, DR or reduced eGFR (p<0.001). Specifically, OPG levels were higher in patients with than in those without CVD (median [IQR]: 11.5 [11.7] vs.10.0 [9.5], p=0.004) or DR (13.3 [14.7] vs.9.7 [8.9], p<0.001), and in patients with an eGFR <45 ml/min/1.73m2 (17.0 [23.5]) compared with an eGFR 45-59 ml/min/1.73m2 (13.2 [13.5]) and ≥60 ml/min/1.73m2 (9.4 [8.7] (p <0.001).To explore potential mechanisms for the association of vascular complications with OPG and/or OPN, we assessed insulin-mediated eNOS activity in bovine aortic endothelial cells cultures (BAECs) with OPG or OPN in the presence of insulin. OPG, but not OPN, inhibited insulin-induced eNOS activation in a dose-dependent manner. Additionally, OPG inhibited insulin-induced activation of both pERK and pAKT in BAECs. In summary, our results show that OPG associates with vascular disease in diabetes and suggest that this effect is mediated by endothelial insulin-resistance and reduced insulin-induced NO production. Disclosure E. Maddaloni: Consultant; Self; Merck KGaA. Speaker’s Bureau; Self; Abbott, AstraZeneca, Pikdare. K. Park: None. M. Di Guida: None. L. Coraggio: None. C. Luordi: None. L. D’Onofrio: None. M.G. Baroni: None. M.G. Cavallo: None. P. D’Angelo: None. S. De Cosmo: None. F. Leonetti: None. S. Morano: None. L. Morviducci: None. P. Pozzilli: Advisory Panel; Self; Abbott, AstraZeneca, Eli Lilly and Company. Research Support; Self; Medtronic, Sanofi. S. Prudente: None. G. Pugliese: Advisory Panel; Self; Novo Nordisk A/S. Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck Sharp & Dohme Corp., Mundipharma International, Sanofi-Aventis, Sigma-tau, Takeda Pharmaceutical Company Limited. Other Relationship; Self; Laboratori Guidotti. V. Trischitta: None. R.R. Holman: Advisory Panel; Self; Merck Sharp & Dohme Corp., Novartis AG, Novo Nordisk A/S. Research Support; Self; AstraZeneca, Bayer AG, Merck Sharp & Dohme Corp. G.L. King: Research Support; Self; Janssen Pharmaceuticals, Inc. R. Buzzetti: Advisory Panel; Self; Sanofi. Speaker’s Bureau; Self; AstraZeneca, Lilly Diabetes, Merck Sharp & Dohme Corp. Funding European Foundation for the Study of Diabetes; AstraZeneca (MS 2017_2); Società Italiana di Diabetologia

Lytic cycle of Besnoitia besnoiti tachyzoites displays similar features in primary bovine endothelial cells and fibroblasts

BackgroundBovine besnoitiosis, caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that continues to spread in Europe in the absence of control tools. In this scenario, in vitro culture systems are valuable tools to carry out drug screenings and to unravel host-parasite interactions. However, studies performed in bovine target cells are scarce.MethodsThe objective of the present study was to obtain primary bovine aortic endothelial cells (BAECs) and fibroblast cell cultures, target cells during the acute and the chronic stage of the disease, respectively, from healthy bovine donors. Afterwards, expression of surface (CD31, CD34 and CD44) and intracellular markers (vimentin and cytokeratin) was studied to characterize cell populations by flow cytometry. Next, the lytic cycle of B. besnoiti tachyzoites was studied in both target cells. Invasion rates (IRs) were determined by immunofluorescence at several time points post-infection, and proliferation kinetics were studied by quantitative PCR (qPCR). Finally, the influence of bovine viral diarrhea virus (BVDV) co-infection on the host cell machinery, and consequently on B. besnoiti invasion and proliferation, was investigated in BAECs.ResultsMorphology and cytometry results confirmed the endothelial and fibroblast origins. CD31 was the surface marker that best discriminated between BAECs and fibroblasts, since fibroblasts lacked CD31 labelling. Expression of CD34 was weak in low-passage BAECs and absent in high-passage BAECs and fibroblasts. Positive labelling for CD44, vimentin and cytokeratin was observed in both BAECs and fibroblasts. Regarding the lytic cycle of the parasite, although low invasion rates (approximately 3–4%) were found in both cell culture systems, more invasion was observed in BAECs at 24 and 72 hpi. The proliferation kinetics did not differ between BAECs and fibroblasts. BVDV infection favoured early Besnoitia invasion but there was no difference in tachyzoite yields observed in BVDV-BAECs compared to BAECs.ConclusionsWe have generated and characterized two novel standardized in vitro models for Besnoitia besnoiti infection based on bovine primary target BAECs and fibroblasts, and have shown the relevance of BVDV coinfections, which should be considered in further studies with other cattle pathogens.

Death receptor 3 mediates TNFSF15- and TNFα-induced endothelial cell apoptosis

Tumor necrosis factor superfamily 15 (TNFSF15) suppresses angiogenesis by specifically inducing apoptosis in proliferating endothelial cells. Death receptor 3 (DR3), a member of the TNF receptor superfamily (TNFRSF25), has been identified as a receptor for TNFSF15 to activate T cells. It is unclear, however, whether DR3 mediates TNFSF15 activity on endothelial cells. Here we show that siRNA-mediated knockdown of DR3 in an in vivo Matrigel angiogenesis assay, or in adult bovine aortic endothelial (ABAE) cell cultures, leads to resistance of endothelial cells to TNFSF15-induced apoptosis. Interestingly, DR3-depleted cells also exhibited markedly diminished responsiveness to TNFα cytotoxicity, even though DR3 is not a receptor for TNFα. Treatment of the cells with either TNFSF15 siRNA or a TNFSF15-neutralizing antibody, 4-3H, also results in a significant inhibition of TNFα-induced apoptosis. Mechanistically, DR3 siRNA treatment gives rise to an increase of ERK1/2 MAPK activity, and up-regulation of the anti-apoptotic proteins c-FLIP and Bcl-2, thus strengthening apoptosis-resisting potential in the cells. These findings indicate that DR3 mediates TNFSF15-induced endothelial cell apoptosis, and that up-regulation of TNFSF15 expression stimulated by TNFα is partly but significantly responsible for TNFα-induced apoptosis in endothelial cells.

A Catalytic Role of Heparin within the Extracellular Matrix

We investigated the mechanism by which heparin enhances the binding of vascular endothelial growth factor (VEGF) to the extracellular matrix protein fibronectin. In contrast to other systems, where heparin acts as a protein scaffold, we found that heparin functions catalytically to modulate VEGF binding site availability on fibronectin. By measuring the binding of VEGF and heparin to surface-immobilized fibronectin, we show that substoichiometric amounts of heparin exposed cryptic VEGF binding sites within fibronectin that remain available after heparin removal. Measurement of association and dissociation kinetics for heparin binding to fibronectin indicated that the interaction is rapid and transient. We localized the heparin-responsive element to the C-terminal 40-kDa Hep2 domain of fibronectin. A mathematical model of this catalytic process was constructed that supports a mechanism whereby the heparin-induced conformational change in fibronectin is accompanied by release of heparin. Experiments with endothelial extracellular matrix suggest that this process may also occur within biological matrices. These results indicate a novel mechanism whereby heparin catalyzes the conversion of fibronectin to an open conformation by transiently interacting with fibronectin and progressively hopping from molecule to molecule. Catalytic activation of the extracellular matrix might be an important mechanism for heparin to regulate function during normal and disease states.

Myoferlin Regulates Vascular Endothelial Growth Factor Receptor-2 Stability and Function

Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling.

Micropatterning with aerosols: Application for biomaterials

Adhesion and proliferation behaviors of bovine aortic endothelial cells (BAECs) were investigated on surfaces micropatterned with peptides using a novel approach. This micropatterning technique allows modification of macroscopic three-dimensional (3D) biomaterials surfaces and exploits the semi-random properties of aerosols and the principles of liquid atomization. The possibility to control cell behaviors on polytetrafluoroethylene (PTFE) surfaces tailored with this micropatterning approach was evaluated. CGRGDS and CWQPPRARI peptides were selected for their adhesive, migration and spreading properties. Culture of BAECs on patterned PTFE showed the possibility of modulating cell behaviors. The study showed that CGRGDS spots with a diameter of 10+/-2 microm over a background of CWQPPRARI peptides was the most effective combination to enhance endothelialization of PTFE. This micropatterning technique is innovative, easily adaptable, simple, and rapid for covering large 3D areas.

Lipid rafts mediate H2O2prosurvival effects in cultured endothelial cells

Reactive oxygen species (ROS) generated during pathological events, such as inflammation and ischemia-reperfusion, activates both proapoptotic and antiapoptotic signaling programs in endothelial cells. Because cholesterol-rich, plasma membrane rafts serve as platforms for organizing and integrating signaling transduction processes, we asked whether these membrane regions play a mechanistic role in H2O2-induced responses. Bovine aortic endothelial cell cultures exposed to a 500-microM bolus of H2O2 showed progressive activation of caspase 3 and an increase in the number of TUNEL-positive cells. Pretreatment with either wortmannin or PD 098059 heightened these apoptotic responses, demonstrating that both PI3 kinase/Akt and ERK1/2 serve as signaling mediators to alleviate H2O2 cytotoxic effects. To investigate the role of lipid rafts in these signaling processes, endothelial cells were pretreated with methyl-beta-cyclodextrin (CD) or filipin to ablate raft structures. H2O2-induced phosphorylation of Akt and ERK 1/2 was attenuated, while caspase 3 and the number of TUNEL positive cells was enhanced in CD-pretreated cells exposed to H2O2. Reconstitution of raft domains restored H2O2-induced Akt and ERK1/2 phosphorylation, which was concomitant with reduction of caspase 3 activation and DNA fragmentation. Taken together, our findings suggest that plasma membrane compartments rich in cholesterol participate in signal transduction pathways activated by oxidative stress.

Manganese Enhances Peroxynitrite and Leukotriene E<sub>4</sub> Formation in Bovine Aortic Endothelial Cells Exposed to Arsenic

Long-term exposure to arsenic in drinking water has been linked to cancer and other health effects, including cardiovascular disease. Arsenic in the environment is found in combination with a range of metals that could influence its toxicity. Manganese, in particular, is a metal that is typically found in conjunction with arsenic in contaminated groundwater. Peroxynitrite is a powerful oxidant formed from the reaction between nitric oxide and superoxide anion. Arsenic has been shown to increase the formation of peroxynitrite in bovine aortic endothelial cells (BAECs) and promote the formation of 3-nitrotyrosine (3-NY) in the atherosclerotic plaque of ApoE-/-/LDLr-/- mice. Arsenic exposure also increases leukotriene E4 (LTE4) formation in both the mice and BAECs, an effect that is partially reversed by the addition of Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor. In the present study, we investigated the effect of adding nontoxic concentrations of manganese along with arsenic to BAEC cultures. Manganese increased arsenic toxicity and enhanced peroxynitrite, 3-NY, and LTE4 formation in BAECs. Addition of LNAME reduced 3-NY formation induced by arsenic/manganese mixtures, but in contrast to its effect on arsenic alone, L-NAME actually increased LTE4 synthesis in BAECs treated with the arsenic/manganese combination. Overall, these data suggest that manganese may exacerbate the toxic effects of arsenic on the vascular system.

Calreticulin Destabilizes Glucose Transporter-1 mRNA in Vascular Endothelial and Smooth Muscle Cells Under High-Glucose Conditions

Substrate autoregulation of glucose transporter-1 (GLUT-1) mRNA and protein expression provides vascular endothelial and smooth muscle cells a sensitive mechanism to adapt their rate of glucose transport in response to changing glycemic conditions. Hyperglycemia-induced downregulation of glucose transport is particularly important in protecting these cells against an excessive influx of glucose and consequently increased intracellular protein glycation and generation of free radicals; both are detrimental in the development of vascular disease in diabetes. We aimed to investigate the molecular mechanism of high glucose-induced downregulation of GLUT-1 mRNA expression in primary bovine aortic vascular endothelial (VEC) and smooth muscle (VSMC) cell cultures. Using RNA mobility shift, UV cross-linking, and in vitro degradation assays, followed by mass-spectrometric analysis, we identified calreticulin as a specific destabilizing trans-acting factor that binds to a 10-nucleotide cis-acting element (CAE(2181-2190)) in the 3'-untranslated region of GLUT-1 mRNA. Pure calreticulin accelerated the rate of GLUT-1 mRNA-probe degradation in vitro, whereas overexpression of calreticulin in vascular cells decreased significantly the total cell content of GLUT-1 mRNA and protein. The expression of calreticulin was augmented in vascular cells exposed to high glucose in comparison with low-glucose conditions. Similarly, increased expression of calreticulin was observed in aortae of diabetic Psammomys obesus in comparison with normoglycemic controls. These data suggest that CAE(2181-2190)-calreticulin complex, which is formed in VSMC and VEC exposed to hyperglycemic conditions, renders GLUT-1 mRNA susceptible to degradation. This interaction underlies the process of downregulation of glucose transport in vascular cells under high-glucose conditions.

Culture of large vessel endothelial cells on floating collagen gels promotes a phenotype characteristic of endothelium in vivo

The vascular endothelium in vivo is a remarkably quiescent cell layer that displays a highly differentiated and tissue-specific phenotype. Once established in culture, endothelial cells (EC) are phenotypically different from their in situ counterparts, displaying altered gene expression, increased mitotic index, and decreased cell density. To determine whether manipulating the microenvironment of cells in vitro would lead to a more differentiated phenotype, we cultured bovine aortic EC on floating collagen gels. EC cultured to confluence on floating gels for 24 or 48 hr display mitotic indices nearly identical to those of quiescent endothelium in vivo, nearly two log orders lower than that of EC cultured to confluence on plastic, and cell density on floating gels also resembles that observed for endothelium in vivo. Culture of EC on floating gels leads to decreased expression of platelet-derived growth factor-B, fibronectin, and fibronectin isoform ED-B, and increased levels of connexin40, relative to cells cultured on plastic. We conclude that culture of bovine aortic EC under standard culture conditions results in a phenotype reminiscent of development and/or wound healing, and that culturing them on a floating collagen gel leads to a more differentiated phenotype, reminiscent of that observed for large vessel EC in vivo.

Bovine aortic endothelial cells are susceptible to hantavirus infection; a new aspect in hantavirus ecology

Hantaviruses are enveloped RNA viruses that belong to the family Bunyaviridae. They are the causative agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Hantaviruses show a worldwide distribution with specific rodent species as natural hosts. It is known that rodents can transmit the virus via feces, urine, saliva, or bites to humans. Additionally, antibodies against different hantaviruses were also found in domestic animals, For example, Danes et al. documented hantavirus-specific IgG titers in 2% of examined cattle [Ceskoslov. Epidemiol. Mikrobiol. Imunol. 41 (1992) 15].In order to clarify the possibility of a nonrodent and nonhuman hantavirus infection, the susceptibility of bovine aortic endothelial cells (BAEC) to Hantavirus serotype Puumala infection was investigated. The hantaviral nucleocapsid protein was detected in 95% of infected BAEC at the fourth cell culture passage 12 weeks after initial infection by immunofluorescence assay (IFA). The presence of Puumala virus (PUU) nucleocapsid protein and the viral glycoproteins G1 and G2 in infected cells were additionally confirmed by Western blot analysis. The viral RNA genome was identified in infected BAEC cultures and in cell-free culture medium at the fourth passage by reverse transcription polymerase chain reaction (RT-PCR), verified by cDNA nucleotide sequence analysis, showing a 98–100% homology to the input virus. The infected BAEC cultures were shown to express αVβ3-integrin surface receptors that are known to mediate virus entry in human cells and revealed no major cytopathic effects (CPEs) as assayed by immunofluorescence staining of the cytoskeletal components actin and microtubules.In the present study, we documented for the first time that a nonrodent and nonhuman aortic endothelial cell culture of bovine origin (BAEC) can be efficiently infected with a hantavirus. This finding is of particular importance because it adds new aspects to questions dealing with host species barrier, viral reservoir, virus transmission, and ecology of hantaviruses.

Low-density lipoprotein in hypercholesterolemic human plasma induces vascular endothelial cell apoptosis by inhibiting fibroblast growth factor 2 transcription.

Apoptosis of vascular endothelial cells (ECs) can be induced in vitro by experimentally modified LDL. Description of proapoptotic circulating lipoproteins may significantly enhance understanding of atherothrombosis pathophysiology. Fast protein liquid chromatography of LDL samples from 7 asymptomatic, hypercholesterolemic patients yielded subfractions L1-L5 in increasing electronegativity. L4 and L5 were not detectable or collectible in normolipidemic samples. In bovine aortic EC cultures, L5 induced marked apoptosis and L4 had a mild effect, whereas hypercholesterolemic or normolipidemic L1-L3 had negligible effects. Compared with copper-oxidized LDL, L5 was only mildly oxidized, although its propensity to form conjugated dienes in response to copper exceeded that of other subfractions. L5-induced apoptosis was associated with suppressed fibroblast growth factor 2 (FGF-2) transcription, as assessed by nuclear run-on analysis. Degrading platelet-activating factor (PAF)-like lipids in L5 by a recombinant PAF acetylhydrolase prevented both FGF-2 downregulation and apoptosis. Furthermore, the ability of L5 lipid extract to induce calcium influx into neutrophils was lost after pretreatment of the extract with PAF acetylhydrolase. FGF-2 supplementation, PAF receptor (PAFR) blockade with WEB-2086, and inactivation of PAFR-coupled Gi protein with pertussis toxin all effectively attenuated L5-induced apoptosis. Our findings indicate that a highly electronegative, mildly oxidized LDL subfraction present in human hypercholesterolemic but not normolipidemic plasma can induce apoptosis in cultured ECs. The evidence that a freshly isolated LDL species modulates transcription of FGF-2 may provide a physiological insight into the mechanism of vascular EC apoptosis in hypercholesterolemia.

Angiostatin: a negative regulator of endothelial-dependent vasodilation.

Angiostatin is known to inhibit certain aspects of endothelial function, eg, angiogenesis. Here we investigated the effects of angiostatin on another aspect of endothelial function, vasodilation, and examined mechanisms of inhibition--namely, association of heat-shock protein 90 (hsp90) with endothelial nitric oxide synthase (eNOS) and endothelial generation of nitric oxide (*NO) and superoxide anion (O2-). This avenue of investigation was based on recent reports suggesting that hsp90 modulates NOS production of *NO and O2-. Effects of angiostatin on vasodilation were determined in arterioles with the use of videomicroscopy in response to endothelium- and *NO-dependent vasodilators, acetylcholine (ACh) and vascular endothelial growth factor (VEGF), and an endothelium-independent agonist, papaverine. Association of hsp90 with eNOS was determined in rat aortas and bovine aortic endothelial cells (BAECs). Effects of angiostatin on *NO and O2- generation by BAECs were determined by ozone chemiluminescence and superoxide dismutase (SOD)--inhibitable ferricytochrome c reduction, respectively. Angiostatin impaired vasodilation mediated by ACh and VEGF but not papaverine. Pretreating arterioles with polyethylene glycolated--SOD (PEG-SOD) improved vasodilation to ACh and VEGF. Angiostatin decreased the association of hsp90 with eNOS in aortas and BAEC cultures and increased O2- generation in stimulated BAECs by an Lgamma-nitroargininemethylester (L-NAME)--inhibitable mechanism. These data indicate angiostatin alters endothelial function by allowing eNOS to generate O2- on activation. Such changes in enzyme function begin to explain, in part, why angiostatin is antiangiogenic and impairs endothelium-dependent vasodilation.

Requirement for GSH in recycling of ascorbic acid in endothelial cells

Ascorbic acid may be involved in the defense against oxidant stress in endothelial cells. Such a role requires that the cells effectively recycle the vitamin from its oxidized forms. In this work, we studied the ability of cultured bovine aortic endothelial cells (BAECs) to take up and reduce dehydroascorbic acid (DHA) to ascorbate, as well as the dependence of ascorbate recycling on intracellular GSH. BAECs took up and reduced DHA to ascorbate much more readily than they took up ascorbate. Although BAECs in culture did not contain ascorbate, ascorbate accumulated to concentrations of 2–3 mM in BAECs following incubation with 400 μM DHA. Extracellular ferricyanide oxidized intracellular ascorbate, which was recycled by the cells. Reduction of DHA, either when added to the cells or when generated in response to ferricyanide, caused significant decreases in intracellular GSH concentrations. Depletion of intracellular GSH with 1-chloro-2,4-dinitrobenzene, diethylmaleate, and diamide almost abolished the ability of the cells to reduce DHA to ascorbate. DHA reduction by thioredoxin reductase was evident in dialyzed cell extracts, but occurred at rates far lower than direct GSH reduction of DHA. These results suggest that maximal rates of DHA reduction, and thus recycling of ascorbate from DHA, are dependent upon GSH in these cells.

Effects of homocysteine on the binding of extracellular-superoxide dismutase to the endothelial cell surface

Homocysteine is known to be a risk factor for several vascular diseases. Previously, we found a significant association between plasma homocysteine and plasma extracellular-superoxide dismutase (EC-SOD) levels. The binding of EC-SOD to human and bovine aortic endothelial cell cultures showed significant decreases after incubation with 10 μM homocysteine, whereas the expression of EC-SOD in fibroblast cell cultures was inhibited with a high concentration (1 mM) of homocysteine. Furthermore, binding of EC-SOD to heparin immobilized on plates was decreased with homocysteine. These observations suggested that homocysteine decreases the binding of EC-SOD to vascular endothelial cell surfaces by degradation of endothelial heparan sulfate proteoglycan, which results in a loss of the ability to protect endothelial cell surfaces from oxidative stress.

5-hydroxytryptamine evokes endothelial nitric oxide synthase activation in bovine aortic endothelial cell cultures.

Activation of endothelial nitric oxide synthase (eNOS) results in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells. The goal of this project was to address the possibility that 5-hydroxytryptamine (5-HT) stimulates eNOS activity in bovine aortic endothelial cell (BAEC) cultures. Here, we tested the hypothesis that 5-HT receptors mediate eNOS activation by measuring agonist-stimulated [3H]L-citrulline ([3H]L-Cit) formation in BAEC cultures. We found that 5-HT stimulated the conversion of [3H]L-arginine ([3H]L-Arg) to [3H]L-Cit, indicating eNOS activation. The high affinity 5-HT1B receptor agonist, 5-nonyloxytryptamine (5-NOT)-stimulated [3H]L-Cit turnover responses were concentration-(0.01 nM to 100 microM) and time-dependent. Maximal responses were observed within 10 min following agonist exposures. These responses were effectively blocked by the 5-HT1B receptor antagonist, isamoltane, the 5-HT1B/5-HT2 receptor antagonist, methiothepin, and the eNOS selective antagonists (0.01-10 microM): L-Nomega -monomethyl-L-arginine (L-NMMA) and L-N omega-iminoethyl-L-ornithine (L-NIO). Pretreatment of BAEC cultures with pertussis toxin (PTX; 1-100 ng/ml) for 16 hr resulted in significant inhibition of the agonist-stimulated eNOS activity, indicating the involvement of Gi proteins. These findings lend evidence of a 5-HT1B receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT. Further investigation is needed to determine the role of other vascular 5-HT receptors in the stimulation of eNOS activity.

Ethanol Increases Endothelial Nitric Oxide Production through Modulation of Nitric Oxide Synthase Expression

Moderate alcohol consumption has been shown to reduce the risk of ischemic heart disease potentially through its effect on specific endothelial-derived compounds. We tested the hypothesis that ethanol increases the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production in bovine aortic endothelial cells (BAEC). Primary cultures of BAEC grown to confluence under standard conditions were treated 3-6 h with 0.1% ethanol in the presence of indomethacin. Ethanol induced a significant increase in both basal and stimulated NO production as determined by chemiluminescence method. This effect was accompanied by a rapid increase of eNOS protein and mRNA expression levels. eNOS mRNA increased two-fold within 3 h and gradually declined, but the increased levels of mRNA persisted for >24 h. A similar increase of eNOS expression was observed in human umbilical endothelial cells exposed to ethanol. These results demonstrate that ethanol augments both basal and stimulated NO production and that this effect is associated with increased eNOS protein and mRNA expression levels. The data are consistent with the hypothesis that the reduced incidence of ischemic heart disease associated with alcohol may be related, at least in part, to the modulation of vascular endothelial cell production of NO.

Aminothiol WR-1065 protects endothelial cell morphology against alterations induced by lipopolysaccharide.

In septic patients, lipopolysaccharide (LPS) damages the vascular endothelium, which manifests as tissue edema and impaired healing. This pathology occurs when LPS distorts endothelial cell morphology partly by generating free radicals. A radioprotector that scavenges free radicals, the aminothiol WR-1065 ([N-2-mercaptoethyl]-1-3-diaminopropane) was found in a prior study to normalize the morphology of irradiated endothelial cells (Mooteri SN, Podolski JL, Drab EA, et al: Radiat Res 145:217-224, 1996). The aim of this study was to determine whether WR-1065 also normalized endothelial cell morphology following exposure to LPS. For this aim, portions of bovine aortic endothelial cell cultures were denuded and exposed to LPS at 1 ng/mL. After 30 min, the apical membrane expressed increased integrin receptor to fibronectin, alpha5beta1. After 5 h, the morphology of the cells at the leading edge was distorted, and cell-cell contact was lessened. Also, filamentous actin-containing stress fibers were dissipated; however, filamentous actin content per cell was unchanged. Treatment with 2 mM WR-1065 for 2 h prior to LPS exposure attenuated the increased expression of alpha5beta1 and promoted cell-cell contact in the migrating endothelial cells. WR-1065 also promoted the retention of stress fibers and actin cytoskeletal shape in cells treated with LPS. Thus, LPS distorted endothelial cell morphology after increasing apical membrane expression of alpha5beta1 and dissipating stress fibers, effects prevented by WR-1065.

Inhibition of lipopolysaccharide (LPS)-induced endothelial cytotoxicity by selected flavonoids.

The cytotoxic effect of lipopolysaccharide (LPS) was examined on bovine aortic endothelial cell proliferation in vitro. This LPS-induced cytotoxicity (IC50 = 22 ng/ml) was attenuated by selected flavonoids, such as catechin, myricetin, quercetin, hesperitin, and rutin. No inhibitory effect was induced by flavones, morin, kaempferol, chrysin and naringin. The isoflavone genistein, and the synthetic tyrphostin B46, both known inhibitors of tyrosine kinases, also attenuated the toxic LPS effect. The hypothesis was framed that the inhibition of LPS-induced cytotoxicity in bovine aortic endothelial cell cultures by flavonoids could be mediated via inhibition of specific tyrosine kinases.

Altered Expression of Cyclic Nucleotide Phosphodiesterase Isozymes during Culture of Aortic Endothelial Cells

Primary cultures of bovine aortic endothelial cells (BAEC) express cyclic nucleotide phosphodiesterase (CN PDE) isozymes of the PDE2, PDE4 and PDE5 gene families. We report here that the isozyme profiles of CN PDE and the amounts of each vary with the passage number of BAEC cultures. Characterization by anion-exchange chromatography and pharmacological criteria were used to study CN PDE in early (4–6), intermediate (6–10), and late (>17) passages of purified BAEC. PDE2 and a minor fraction of PDE5 accounted for cyclic GMP hydrolysis in early passages, but both isozymes were lost with cell passage. Cyclic AMP was hydrolyzed by both PDE2 and PDE4 isozymes in early passage endothelial cells, but PDE4 was increased dramatically in higher passage cells. Also appearing in the higher passage cells were prominent PDE1 and minor PDE3 activities. The ratios of cytosolic to particulate activities were similar at all passages. BAEC PDE isoforms in intact cells assessed by [ 3H]-adenine prelabeling showed that atriopeptin II decreased isoproterenol-induced cyclic AMP accumulation in early but not later passage cells, consistent with the loss of PDE2 expression. Enhancement of isoproterenol-induced cyclic AMP accumulation by rolipram, a PDE4 inhibitor, was also greatly diminished during culture passages. Changes in CN PDE isoform expression and consequent cyclic AMP turnover validate the importance of considering cell passage number when cultures of BAEC are used to study the regulation of endothelial cell cyclic nucleotide metabolism and processes mediated by cyclic nucleotides in this model system.